It is well established that ras oncogenes play a central role in the pathogenesis of a wide variety of human malignancies. Ras proteins are essential components of receptor-mediated signal transduction pathways regulating cell growth and differentiation. The long term objective of this research project is to define the pathways by which receptors act through Ras to control cell growth and differentiation. This will provide a necessary perspective from which to achieve an understanding of the regulation of cell growth by extracellular signals. We have recently shown that the guanine nucleotide exchange factor Sos mediates the coupling of receptor tyrosine kinases to Ras activation. The goal of the proposed studies is to define the regulation of Ras activation by Sos both at the structural and functional levels. Based on the observation that growth factors induce the serine/threonine phosphorylation of Sos, the first aim of this project is to determine the functional significance of this phosphorylation. To this end, biochemical studies will be carried out to characterize the growth factor-stimulated sites of Sos phosphorylation. Based on this analysis, Sos mutants lacking the phosphorylation sites will be generated and used to study the physiological relevance of Sos mutants lacking the phosphorylation sites will be generated and used to study the physiological relevance of Sos phosphorylation. With the goal of gaining a broader understanding of the regulatory role of Sos proteins, the second aim of this project is directed at the identification of inter- and intra-molecular interactions required for Sos function. Mutagenesis studies will be used in combination with biochemical and functional assays to define the regulatory activities of various Sos domains. Using protein-protein interaction screens, novel Sos binding proteins will be identified and cloned. Lastly, the third aim of this proposal is to elucidate the structural basis of Sos-mediated guanine nucleotide exchange activity. X-ray crystallography and molecular molding will be used to produce a three dimensional structure of the catalytic domain of Sos. Structural determinants that are critical for the interaction of Sos catalytic domain with Ras will be identified by site directed mutagenesis and biochemical analysis. Overall, experiments proposed in this application should serve the purpose of deepening our understanding of a critical regulatory step in the Ras signaling pathway.